O-linked N-acetylglucosamine transferase (OGT) regulates pancreatic α-cell function in mice.

The nutrient sensor O-GlcNAc transferase (OGT) catalyzes posttranslational addition of O-GlcNAc onto target proteins, influencing signaling pathways in response to cellular nutrient levels. OGT is highly expressed in pancreatic glucagon-secreting cells (α-cells), which secrete glucagon in response to hypoglycemia. The objective of this study was to determine whether OGT is necessary for the regulation of α-cell mass and function in vivo. We utilized genetic manipulation to produce two α-cell specific OGT-knockout models: a constitutive glucagon-Cre (αOGTKO) and an inducible glucagon-Cre (i-αOGTKO), which effectively delete OGT in α-cells. Using approaches including immunoblotting, immunofluorescent imaging, and metabolic phenotyping in vivo, we provide the first insight on the role of O-GlcNAcylation in α-cell mass and function. αOGTKO mice demonstrated normal glucose tolerance and insulin sensitivity but displayed significantly lower glucagon levels during both fed and fasted states. αOGTKO mice exhibited significantly lower α-cell glucagon content and α-cell mass at 6 months of age. In fasting, αOGTKO mice showed impaired pyruvate stimulated gluconeogenesis in vivo and reduced glucagon secretion in vitro. i-αOGTKO mice showed similarly reduced blood glucagon levels, defective in vitro glucagon secretion, and normal α-cell mass. Interestingly, both αOGTKO and i-αOGTKO mice had no deficiency in maintaining blood glucose homeostasis under fed or fasting conditions, despite impairment in α-cell mass and function, and glucagon content. In conclusion, these studies provide a first look at the role of OGT signaling in the α-cell, its effect on α-cell mass, and its importance in regulating glucagon secretion in hypoglycemic conditions.

Deficiency of glucagon gene-derived peptides induces peripheral polyneuropathy in mice.

Although diabetic polyneuropathy (DPN) is the commonest diabetic complication, its pathology remains to be clarified. As previous papers have suggested the neuroprotective effects of glucagon-like peptide-1 in DPN, the current study investigated the physiological indispensability of glucagon gene-derived peptides (GCGDPs) including glucagon-like peptide-1 in the peripheral nervous system (PNS). Neurological functions and neuropathological changes of GCGDP deficient (gcg-/-) mice were examined. The gcg-/- mice showed tactile allodynia and thermal hyperalgesia at 12-18 weeks old, followed by tactile and thermal hypoalgesia at 36 weeks old. Nerve conduction studies revealed a decrease in sensory nerve conduction velocity at 36 weeks old. Pathological findings showed a decrease in intraepidermal nerve fiber densities. Electron microscopy revealed a decrease in circularity and an increase in g-ratio of myelinated fibers and a decrease of unmyelinated fibers in the sural nerves of the gcg-/- mice. Effects of glucagon on neurite outgrowth were examined using an ex vivo culture of dorsal root ganglia. A supraphysiological concentration of glucagon promoted neurite outgrowth. In conclusion, the mice with deficiency of GCGDPs developed peripheral neuropathy with age. Furthermore, glucagon might have neuroprotective effects on the PNS of mice. GCGDPs might be involved in the pathology of DPN.

Hepatic Glucagon Signaling Regulates PCSK9 and Low-Density Lipoprotein Cholesterol.

RATIONALE: Glucagon is a key hormone that regulates the adaptive metabolic responses to fasting. In addition to maintaining glucose homeostasis, glucagon participates in the regulation of cholesterol metabolism; however, the molecular pathways underlying this effect are incompletely understood. OBJECTIVE: We sought to determine the role of hepatic Gcgr (glucagon receptor) signaling in plasma cholesterol regulation and identify its underlying molecular mechanisms. METHODS AND RESULTS: We show that Gcgr signaling plays an essential role in LDL-C (low-density lipoprotein cholesterol) homeostasis through regulating the PCSK9 (proprotein convertase subtilisin/kexin type 9) levels. Silencing of hepatic Gcgr or inhibition of glucagon action increased hepatic and plasma PCSK9 and resulted in lower LDLR (LDL receptor) protein and increased plasma LDL-C. Conversely, treatment of wild-type (WT) mice with glucagon lowered LDL-C levels, whereas this response was abrogated in Pcsk9-/- and Ldlr-/- mice. Our gain- and loss-of-function studies identified Epac2 (exchange protein activated by cAMP-2) and Rap1 (Ras-related protein-1) as the downstream mediators of glucagon's action on PCSK9 homeostasis. Moreover, mechanistic studies revealed that glucagon affected the half-life of PCSK9 protein without changing the level of its mRNA, indicating that Gcgr signaling regulates PCSK9 degradation. CONCLUSIONS: These findings provide novel insights into the molecular interplay between hepatic glucagon signaling and lipid metabolism and describe a new posttranscriptional mechanism of PCSK9 regulation.

Conventional insulin pump therapy in two neonatal diabetes patients harboring the homozygous PTF1A enhancer mutation: Need for a novel approach for the management of neonatal diabetes.

Kurnaz E, Aycan Z, Yildirim N, Çetinkaya S. Conventional insulin pump therapy in two neonatal diabetes patients harboring the homozygous PTF1A enhancer mutation: Need for a novel approach for the management of neonatal diabetes. Turk J Pediatr 2017; 59: 458-462. The enhancer of PTF1A mutation causes developmental defects of the pancreas. This condition can result in insulin-requiring diabetes and exocrine pancreatic insufficiency. We report two patients with diabetes mellitus harboring the homozygous PTF1A enhancer mutation. The patients had hyperglycemia in the first month of life and were started with subcutaneous insulin injections with NPH insulin. When blood glucose (BG) exceeded 250 mg/dl, a conservative dose of rapid-acting insulin was administered to restore BG to the target range. In cases with documented poor control (persistent hypoglycemia and rebound hyperglycemia), it was decided that the baby would benefit from a continuous subcutaneous insulin infusion pump. But our experience shows that wide fluctuations in BG concentrations despite the strict follow-up was probably due to the absence of circulating glucagon. Further treatment options would overcome this problem, especially for children with pancreas agenesis. We could say theoretically that using diluted insulin, a sensor-augmented insulin pump system and single-hormone (insulin alone) and dual-hormone (insulin and glucagon) artificial pancreas systems may mitigate the severity of hypogylcemia.

Metabolic impact of glucagon deficiency.

Multiple bioactive peptides are produced from proglucagon encoded by glucagon gene (Gcg). Glucagon is produced in islet α-cells through processing by prohormone convertase 2 (Pcsk2) and exerts its action through the glucagon receptor (Gcgr). Although it is difficult to produce a genetic model that harbours isolated glucagon deficiency without affecting the production of other peptides derived from proglucagon, three different animal models that harbour deficiencies in glucagon signalling have been generated by gene targeting strategy. Although both Pcsk2(-/-) and Gcgr(-/-) mice display lower blood glucose levels, homozygous glucagon-GFP knock-in mice (Gcg(gfp/gfp) ) display normoglycaemia despite complete glucagon deficiency. In Gcg(gfp/gfp) mice, the metabolic impact of glucagon deficiency is probably ameliorated by lower plasma insulin levels and glucagon-independent mechanisms that maintain gluconeogenesis. As both Pcsk2(-/-) and Gcgr(-/-) mice exhibit increased production of glucagon-like peptide-1 (GLP-1), which is absent in Gcg(gfp/gfp), GLP-1 is the likely cause of the difference in metabolic impact of glucagon deficiency in these animal models. Although all the three models display islet 'α'-cell hyperplasia, the mechanisms involved remain to be elucidated. Studies using Pcsk2(-/-), Gcgr(-/-) and Gcg(gfp/gfp) mice, especially in combination with α-cell ablation models such as pancreas-specific aristaless-related homeobox (ARX) knockout mice, should further clarify the physiological and pathological roles of glucagon in the regulation of metabolism and the control of islet cell differentiation and proliferation.

Glucagon deficiency reduces hepatic glucose production and improves glucose tolerance in adult mice.

The major role of glucagon is to promote hepatic gluconeogenesis and glycogenolysis to raise blood glucose levels during hypoglycemic conditions. Several animal models have been established to examine the in vivo function of glucagon in the liver through attenuation of glucagon via glucagon receptor knockout animals and pharmacological interventions. To investigate the consequences of glucagon loss to hepatic glucose production and glucose homeostasis, we derived mice with a pancreas specific ablation of the alpha-cell transcription factor, Arx, resulting in a complete loss of the glucagon-producing pancreatic alpha-cell. Using this model, we found that glucagon is not required for the general health of mice but is essential for total hepatic glucose production. Our data clarifies the importance of glucagon during the regulation of fasting and postprandial glucose homeostasis.

The ectopic expression of Pax4 in the mouse pancreas converts progenitor cells into alpha and subsequently beta cells.

We have previously reported that the loss of Arx and/or Pax4 gene activity leads to a shift in the fate of the different endocrine cell subtypes in the mouse pancreas, without affecting the total endocrine cell numbers. Here, we conditionally and ectopically express Pax4 using different cell-specific promoters and demonstrate that Pax4 forces endocrine precursor cells, as well as mature alpha cells, to adopt a beta cell destiny. This results in a glucagon deficiency that provokes a compensatory and continuous glucagon+ cell neogenesis requiring the re-expression of the proendocrine gene Ngn3. However, the newly formed alpha cells fail to correct the hypoglucagonemia since they subsequently acquire a beta cell phenotype upon Pax4 ectopic expression. Notably, this cycle of neogenesis and redifferentiation caused by ectopic expression of Pax4 in alpha cells is capable of restoring a functional beta cell mass and curing diabetes in animals that have been chemically depleted of beta cells.

[Hypoglycemia as a limiting factor in the management of type 1 diabetes]. / Hipoglicemia como fator complicador no tratamento do diabetes melito tipo 1.

Type 1 diabetic patients frequently present hypoglycemic episodes during their insulinotherapy, which, besides the discomfort and constrains does not allow the ideal glycemic control. Further, hypoglycemic events lead to the deficiency of the counter-regulation mechanisms in the subsequent episode, with a decrease in the release of epinephrine and the symptoms of warming, with great risk of severe hypoglycemia. The occurrence of hypoglycemia during some risky activities, specially driving, could result in accidents with the patient and /or third parts including property damage, stressing here the need to advise diabetics against having the necessary caution while driving. Generally the connective recovery is total after correcting a hypoglycemic coma. However when these episodes are repetitive, particularly in children, they could result in definitive cognitive disturbances. Hypoglycemic events without a warning signal (hypoglycemic unawareness) are difficult to reverse, thus it is necessary to prevent their occurrence, adjusting the treatment with glycemic targets, using continuous glucose monitoring at home and teaching them how to have an early recognition of hypoglycemia.

Hipoglicemia como fator complicador no tratamento do diabetes melito tipo 1: [revisão] / Hypoglycemia as a Limiting Factor in the Management of Type 1 Diabetes: [review]

Os portadores de diabetes melito tipo 1 têm, com freqüência, episódios de hipoglicemia durante a insulinoterapia, que, além do desconforto e de proporcionar situações constrangedoras no dia-a-dia, impedem a obtenção do controle glicêmico ideal. Mais ainda, hipoglicemias induzem deficiente mecanismo de contra-regulação em episódio posterior, com diminuição de liberação de adrenalina e dos sintomas de alarme, estabelecendo a síndrome de hipoglicemia associada à insuficiência autonômica. A ocorrência de hipoglicemias durante algumas atividades de risco, em especial a direção veicular, pode resultar acidentes com o paciente e terceiros, além de lesão de propriedade, motivo pelo qual pessoas com diabetes devem ser orientadas quanto aos cuidados na direção de veículos. Em geral, a recuperação neurológica é total após a correção de coma hipoglicêmico. No entanto, quando esses episódios são repetitivos, especialmente em crianças, podem ter como conseqüência distúrbios cognitivos definitivos. A reversão de quadros de hipoglicemia sem sinal de alerta é difícil, devendo-se evitar meticulosamente sua ocorrência, adequando o tratamento, os alvos glicêmicos, utilizando a monitoração domiciliar e fazendo treinamento para o reconhecimento precoce de hipoglicemias.

Type 1 diabetic patients frequently present hypoglycemic episodes during their insulinotherapy, which, besides the discomfort and constrains does not allow the ideal glycemic control. Further, hypoglycemic events lead to the deficiency of the counter-regulation mechanisms in the subsequent episode, with a decrease in the release of epinephrine and the symptoms of warming, with great risk of severe hypoglycemia. The occurrence of hypoglycemia during some risky activities, specially driving, could result in accidents with the patient and /or third parts including property damage, stressing here the need to advise diabetics against having the necessary caution wheli driving. Generally the connective recovery is total after correcting a hypoglycemic coma. However when these episodes are repetitive, particularly in children, they could result in definitive cognitive disturbances. Hypoglycemic events without a warning signal (hypoglycemic unawareness) are difficult to reverse, thus it is necessary to prevent their occurrence, adjusting the treatment with glycemic targets, using continuous glucose monitoring at home and teaching them how to have an early recognition of hypoglycemia.

Glucagon: the effects of its excess and deficiency on insulin action.

AIM: To review the role that glucagon plays in physiology, physiopathology and clinical medicine. DATA SYNTHESIS: Glucagon assays employing specific radioimmunoassay (RIA) techniques are now widely used to characterize pathologic conditions where the effect of the excess or deficiency of glucagon on insulin actions might play a role. Glucagon excess counteracts the action of insulin on glucose metabolism by stimulating glycogenolysis and gluconeogenesis. Aside from glucagon excess in association with glucagonoma, glucagon excess is found in several metabolic disturbances. In diabetes mellitus, hyperglycaemia is the consequence of the glycogenolytic and gluconeogenic effects of glucagon excess occurring in the setting of a relative insulin deficiency (i.e. Type 2 diabetes), whereas excess of glucagon and absent insulin levels are typical features of diabetic ketoacidosis. Although plasma glucagon levels of patients with diabetes are usually increased relative to the prevailing plasma glucose concentrations, it is a paradox that in those patients glucagon levels fail to rise when hypoglycaemia develops. Since glucagon release is considered the primary defence against insulin-induced hypoglycaemia, the defective response of glucagon to hypoglycaemia may favour the development of severe hypoglycaemia. Such defective response to hypoglycaemia in diabetes can be regarded as a condition of selective glucagon deficiency the mechanisms of which remain to be elucidated. CONCLUSION: The most common condition associated with glucagon excess or deficiency is diabetes mellitus. Glucagon excess contributes to hyperglycaemia whereas reduced glucagon response to insulin-induced hypoglycaemia promotes severe hypoglycaemia. It is expected that drugs that are able to reduce glucagon secretion in concert with strategies directed to recover glucagon secretion to hypoglycaemia might contribute to improve the overall glycaemic control in diabetes.

Role of endogenous glucagon-like peptide-1 in islet regeneration after partial pancreatectomy.

A reduction in beta-cell mass is an important causative factor in type 1 and type 2 diabetes. Glucagon-like peptide-1 (GLP-1) and the long-acting agonist exendin 4 (Ex-4) expand beta-cell mass by stimulating neogenesis and proliferation. In the partial pancreatectomy (Ppx) model, exogenous Ex-4 promotes islet regeneration, leading to sustained improvement in glucose tolerance. In this study, we investigate the potential role of endogenous GLP-1 in islet growth. We examined beta-cell mass regeneration after 70% Ppx in mice receiving the GLP-1 antagonist Ex9-39 and in GLP-1R(-/-) mice. In Ex9-39-treated sham-operated mice, persistent fasting hyperglycemia was observed, but beta-cell mass was not diminished. In pancreatectomized mice, persistent glucose intolerance was noted, but this was not further exacerbated by Ex9-39. Accordingly, beta-cell mass recovery of Ppx mice was not impaired by Ex9-39. In contrast, GLP-1R(-/-) CD1 mice showed worse glucose intolerance after Ppx compared with wild-type CD1 Ppx mice, and this correlated with a significant defect in beta-cell mass regeneration. The recovery of beta-cell mass differed markedly in the BALB/c and CD1 control mice, indicating a significant role of genetic background in the regulation of beta-cell mass. These studies point to a role for endogenous GLP-1 in beta-cell regeneration after Ppx in mice.

Deficiency of the intestinal growth factor, glucagon-like peptide 2, in the colon of SCID mice with inflammatory bowel disease induced by transplantation of CD4+ T cells.

BACKGROUND: Glucagon-like peptide 2 (GLP-2) is produced in endocrine L-cells of the intestinal mucosa. Recently, GLP-2 was found to stimulate intestinal mucosal growth. Our objective was to study the content of GLP-2 in the large intestine in a murine model of T-cell-induced inflammatory bowel disease. METHODS: Inflammation was induced by adoptive transfer of CD4+ blast T cells from BALB/c mice to SCID mice. The amount of GLP-2 (1-33) was measured with a specific, NH2-terminally directed radioimmunoassay in tissue extracts from the large intestine of transplanted mice developing colitis and from BALB/c and SCID control mice. RESULTS: In the middle and descending colon segments showing the most severe signs of inflammatory lesions in the CD4+ T-cell-transplanted mice, the amount of GLP-2 was significantly lower than in similar colon segments in both untransplanted SCID mice and normal BALB/c mice (P = 0.0013 and 0.0033). In the descending colon the amount of GLP-2 was 6.7 +/- 1.0 pmol/g protein in the CD4+ transplanted mice compared with 68.4 +/- 20.3 and 42.7 +/- 4.3 in the two groups of control mice. Similar findings were made with regard to the contents of the two other proglucagon-derived intestinal peptides, glicentin and GLP-1. CONCLUSION: The amount of GLP-2 is markedly reduced in the colon of mice with a T-cell-induced inflammatory bowel disease histopathologically resembling both Crohn disease and ulcerative colitis. This observation may provide a pathophysiologic rationale for administration of GLP-2 as a trophic factor in inflammatory bowel disease.

The neuroendocrine protein 7B2 is required for peptide hormone processing in vivo and provides a novel mechanism for pituitary Cushing's disease.

The neuroendocrine protein 7B2 has been implicated in activation of prohormone convertase 2 (PC2), an important neuroendocrine precursor processing endoprotease. To test this hypothesis, we created a null mutation in 7B2 employing a novel transposon-facilitated technique and compared the phenotypes of 7B2 and PC2 nulls. 7B2 null mice have no demonstrable PC2 activity, are deficient in processing islet hormones, and display hypoglycemia, hyperproinsulinemia, and hypoglucagonemia. In contrast to the PC2 null phenotype, these mice show markedly elevated circulating ACTH and corticosterone levels, with adrenocortical expansion. They die before 9 weeks of severe Cushing's syndrome arising from pituitary intermediate lobe ACTH hypersecretion. We conclude that 7B2 is indeed required for activation of PC2 in vivo but has additional important functions in regulating pituitary hormone secretion.

Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats.

The role of glucagon in diabetic hyperglycaemia has been a matter of controversy because of difficulties in the production of selective glucagon deficiency. We developed a high-capacity (40 nmol/ml), high-affinity (0.6 x 10(11) l/mol) monoclonal glucagon antibody (Glu-mAb) and gave i.v. injections (4 ml/kg) to rats in order to study the effect of selective glucagon deficiency on blood glucose. Controls received a mAb against trinitrophenyl. Glu-mAb completely abolished the hyperglycaemic effect of 2.86 nmol/kg glucagon in normal rats (p < 0.05, n = 6). In moderately hyperglycaemic rats injected with streptozotocin as neonates (N-STZ), Glu-mAb abolished a postprandial increase in blood glucose (from 11.2 +/- 0.7 mmol/l to 17.3 +/- 1.8 mmol/l in controls vs 10.5 +/- 0.9 mmol/l to 9.3 +/- 1.0 mmol/l; cross-over: n = 6, p < 0.05). No significant effect of Glu-mAb treatment was observed in more hyperglycaemic N-STZ rats (cross-over, n = 4) and in severely hyperglycaemic rats injected with STZ as adults (n = 6), but after insulin treatment of the latter, at doses partially restoring blood glucose levels (12.7 +/- 4.3 mmol/l), Glu-mAb administration almost normalized blood glucose (maximal difference: 6.0 +/- 3.8 mmol/l; cross-over: n = 5, p < 0.05). In conclusion, our results provide strong additional evidence for the hypothesis that glucagon is involved in the pathogenesis of diabetes. The hormone plays an important role in the development of STZ-diabetic hyperglycaemia, but glucagon neutralization only leads to normoglycaemia in the presence of insulin.

Hypoglycaemia unawareness.

Hypoglycaemia is a severe complication of insulin-dependent diabetes mellitus, especially when it is not preceded by warning signs. A patient unaware of a low blood glucose concentration may not take any remedial action, becomes severely hypoglycaemic and may lapse into a potentially fatal coma. Hypoglycaemic unawareness is associated particularly with good glycaemic control. Hypoglycaemia unawareness was first related to diabetic autonomic neuropathy. Subsequently it emerged that well controlled diabetic patients suffered from hypoglycaemia unawareness more often than poorly controlled patients. Next to autonomic neuropathy and good glycaemic control, the transfer of more slowly resorbed beef and porcine insulin to more rapidly resorbed human insulin has been incriminated as causing an increased incidence of hypoglycaemia unawareness. Reviewing the scientific literature concerning the connection between hypoglycaemia unawareness and the use of human insulin, there is no rationale for supporting the hypothesis that human insulin leads to hypoglycaemia unawareness any longer. While the pathophysiology of hypoglycemia unawareness remains unclear in patients with insulin-dependent diabetes mellitus of long duration, it may well be related to impaired adrenaline secretion. The cause of this impaired adrenaline secretion during hypoglycaemia is unknown and it does not seem to be a result of diabetic autonomic neuropathy.